The long-term objective of this project is to understand the mechanisms by which chromatin is assembled and utilized in the eukaryotic nucleus. The studies of chromatin assembly will be focused on the biochemical and biological functions of ACF. ACF is an ATP-utilizing chromatin assembly factor that consists of two subunits, Acf1 and the ISWI ATPase. In conjunction with a core histone chaperone, ACF mediates the ATP-dependent deposition of histones onto DNA as well as the generation of periodic arrays of nucleosomes. This project will also examine the biochemical activities of Rad54, which is involved in homologous recombination. Rad54 is closely related to the ISWI subunit of ACF, and it is therefore anticipated that Rad54 will have a key role in facilitating homologous recombination in chromatin. The Specific Aims are as follows. 1. Investigate the mechanism of ATP-dependent chromatin assembly in vitro. Biochemical and single-molecule approaches will be employed to analyze ACF-mediated chromatin assembly. In addition, factors that mediate ATP-dependent chromatin assembly in the absence of ACF will be examined. 2. Analyze the function of ACF in vivo in Drosophila. A null mutant allele of acf1 was generated by imprecise excision of a P element. By using the homozygous mutant flies, the effect of Acf1 upon gene expression and chromosome structure will be investigated. The localization and function of Acf1 in cells and in polytene chromosomes will also be examined. 3. Examine the ability of Rad54 to mediate strand pairing in chromatin. The mechanism by which Rad54 and Rad51 catalyze D loop formation in chromatin will be investigated. In addition, native Rad54, which appears to be in a multisubunit complex, will be purified from Drosophila embryos. These studies should contribute to our fundamental understanding of chromatin assembly as well as DNA recombination and repair, and should thus provide critical knowledge that would be applicable to the understanding and treatment of human diseases that involve defects in these processes.